To evaluate the long-term impacts of early-life nutritional manipulations on glucagon secretion and hepatic signalling, thirty-six twin-pregnant ewes during their last trimester were exposed to NORM (fulfilling 100% of daily energy/protein requirements), HIGH (fulfilling 150/110% of daily energy/protein requirements) or LOW (50% of NORM) diets. Twin lambs were assigned after birth to a moderate (CONV) or high-carbohydrate high-fat (HCHF) diet until 6 months. Then, responses in plasma glucagon concentrations and glucagon ratios relative to previously reported values for insulin, glucose and lactate were determined after intravenous bolus injections of glucose or propionate (fed and 2-day fasting state). Hepatic mRNA expressions of glucagon receptor (GCGR), glucose-6-phosphatase (G6PC), phosphoenolpyruvate carboxykinase (PEPCK) and fructose 1,6-biphosphatase (FBP) were also determined in a sub group of autopsied lambs. Expression of GCGR and all three enzymes were supressed by prenatal LOW compared to NORM (except PEPCK) and HIGH (except FBP) nutrition. The postnatal HCHF diet reduced plasma glucagon responses to propionate and hepatic mRNA expression of all genes. In response to propionate, insulin/glucagon ratio was decreased (fasted state), but lactate/glucagon and glucose/glucagon increased in HCHF compared to CONV lambs. In conclusion, prenatal undernutrition and postnatal overnutrition had similar long-term implications and reduced hepatic glucagon signalling. Glucagon secretory responses to propionate were, however, not related to the prenatal nutrition history, but negatively affected by the postnatal obesogenic diet. The pancreatic α-cell compared to β-cells may thus be less sensitive towards late gestation malnutrition, whereas hepatic glucagon signalling appears to be a target of prenatal programming.